As a cofactor for thousands of enzymes, iron is an essential micronutrient. Yet, free iron is toxic because it catalyzes rapid formation of damaging reactive oxygen species. Therefore, homeostasis systems exert tight control on iron levels in all organisms and gene expression is adjusted in response to iron deprivation and iron abundance: expression of at least 100 genes is known to be iron-dependent in Escherichia coli. In humans, disruption of iron homeostasis contributes to severe diseases: iron accumulation in the brain is linked to neurodegenerative diseases, iron overload causes the liver disease hemochromatosis, and iron deficiency leads to anemia and impaired cognitive development. Furthermore, invading bacterial pathogens hijack iron out of human proteins to establish infections. These considerations underscore the essential role of iron homeostasis to human health. Because of a lack of studies examining the total cellular framework for the response to both iron deficiency and iron overload, however, how iron homeostasis is integrated into cell physiology is still unclear. Here, to address this deficiency, a two-pronged genome-wide approach is used consisting of: (1) ribosome profiling to determine how expression of genes changes over time in response to varying iron levels; and (2) genome-wide screens to identify genes that are important for survival under iron overload and iron deficiency. This approach is complemented by biochemical and genetic studies to mechanistically characterize new players in iron homeostasis. Importantly, two model systems will be investigated, the gram-negative bacterium Escherichia coli and human cells, allowing for an informative comparison of the two systems that will be essential to develop antibiotics specifically targeted at bacterial iron homeostasis. This comprehensive study will provide a time-resolved and holistic view of the response to iron and identify new players in iron homeostasis, including post-transcriptional regulators, transport systems to take up iron from different sources, and metabolic pathways that become activated in response to iron. Insight obtained from these experiments will benefit understanding of iron homeostasis diseases and treatment of bacterial infections.